Dimeric cationic lipids

ABSTRACT

The present invention provides novel dimeric cationic lipids. The present invention further provides compositions of these lipids with anionic or polyanionic macromolecules, methods for interfering with protein expression in a cell utilizing these compositions and a kit for preparing the same.

TECHNICAL FIELD

The present invention is directed to dimeric cationic lipid compoundsuseful in lipid aggregates for the delivery of macromolecules intocells.

BACKGROUND AND INTRODUCTION TO THE INVENTION

Some bioactive substances do not need to enter cells to exert theirbiological effect, because they operate either by acting on cellsurfaces through cell surface receptors or by interacting withextracellular components. However, many natural biological molecules andtheir analogues, such as proteins and polynucleotides, or foreignagents, such as synthetic molecules, which are capable of influencingcell function at the subcellular or molecular level are preferablyincorporated within the cell in order to produce their effect. For theseagents the cell membrane presents a selective barrier which may beimpermeable to them.

While these membranes serve a protective function by preventing entry oftoxic substances, they can also prevent passage of potentiallybeneficial therapeutic agents into the body. This protective function isinfluenced by the complex composition of the cell membrane whichincludes phospholipids, glycolipids, cholesterol, and intrinsic andextrinsic proteins, as well as by a variety of cytoplasmic components.Interactions between these structural and cytoplasmic cell componentsand their response to external signals make up transport processesresponsible for the membrane selectivity exhibited within and among celltypes.

Successful intracellular delivery of agents not naturally taken up bycells has been achieved to some extent by exploiting natural deliveryvehicles, such as viruses, that can penetrate a cell's membrane or aretaken up by the cell's natural transport mechanisms or by naturalprocess of intracellular membrane fusion. (Duzgunes, N., SubcellularBiochemistry 11:195-286, 1985).

The membrane barrier may be overcome in the first instance by viralinfection or transduction. Various techniques for introducing the DNA ormRNA precursors of bioactive peptides into cells include the use ofviral vectors, such as recombinant vectors and retroviruses, which havethe inherent ability to penetrate cell membranes. However, the use ofsuch viral agents to integrate exogenous DNA into the chromosomalmaterial of the cell carries a risk of damage to the genome and thepossibility of inducing malignant transformation.

Another aspect of this approach which restricts its use in vivo is thatthe integration of DNA into the genome accomplished by these methodsimplies a loss of control over the expression of the peptide it encodes,so that transitory therapy is difficult to achieve and potentialunwanted side effects of the treatment could be difficult or impossibleto reverse or terminate.

The membrane barrier may also be overcome by associating these agents incomplexes with lipid formulations closely resembling the lipidcomposition of natural cell membranes. These lipids are able to fusewith the cell membranes, and in the process, the associated agents aredelivered intracellularly. The structure of various types of lipidaggregates in formulations vary depending on a variety of factors whichinclude composition and methods of forming the aggregate. Lipidaggregates include, for example, liposomes, unilamellar vesicles,multilamellar vesicles, micelles and the like, and may have particlesizes in the nanometer to micrometer range.

The lipids of these formulations may comprise an amphipathic lipid, suchas the phospholipids of cell membranes, which form hollow lipid vesiclesor liposomes in aqueous systems either spontaneously or by mechanicalagitation. This property can be used to entrap the agent to be deliveredwithin the liposomes. In other applications, the agent of interest canbe incorporated into the lipid vesicle as an intrinsic membranecomponent, rather than entrapped in the hollow aqueous interior.

Liposomes have been utilized as in vivo delivery vehicles and someencouraging results were obtain when this approach was applied tointracellular expression of DNA (Mannino, R. J. and Fould-Fogerite, S.,Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276. 1987;Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R. M. andPapahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and Huang,L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987; however, themethodology has fundamental problems. An important drawback to the useof this type of aggregate as a cell delivery vehicle is that theliposome has a negative charge that reduces the efficiency of binding toa negatively charged target cell surface. Consequently, the liposome isoften taken up by the cell phagocytically. Phagocytized liposomes aredelivered to the lysosomal compartment, where polynucleotides aresubjected to the action of digestive enzymes and degraded, which leadsto low efficiency of expression.

A major advance in this area was the discovery that a positively chargedsynthetic cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),in the form of liposomes, or small vesicles, could interactspontaneously with DNA to form lipid-DNA complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting in both uptake and expression of the DNA(Felgner, P. L. et al. Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 andU.S. Pat. No. 4,897,355 to Eppstein, D. et al.). Others havesuccessfully used a DOTMA analogue,1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) in combinationwith a phospholipid to form DNA-complexing vesicles. Lipofectin™(Bathesda Research Laboratories, Gaithersburg, Md.) is an effectiveagent for the delivery of highly anionic polynucleotides into livingtissue culture cells that comprises positively charged DOTMA liposomeswhich interact spontaneously with negatively charged polynucleotides toform complexes. When enough positively charged liposomes are used, thenet charge on the resulting complexes is also positive. Positivelycharged complexes prepared in this way spontaneously attach tonegatively charged cell surfaces, fuse with the plasma membrane, andefficiently deliver functional polynuleotide into, for example, tissueculture cells.

Although the use of known cationic lipids overcomes some of the problemsassociated with conventional liposome technology for polynucleotidedelivery in vitro, problems related to both in vitro and in vivoapplications remain. Cationic lipids such as DOTMA are toxic to tissueculture cells and are expected to accumulate in the body due to theirpoorly metabolized ether bonds.

Another commercially available cationic lipid,1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane ("DOTAP") differs fromDOTMA in that the oleoyl moieties are linked by ester, rather than etherlinkages. However, DOTAP is reported to be more readily degraded bytarget cells leading to low efficiency of delivery.

Other reported cationic lipid compounds include those which have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide("DOGS") and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide("DPPES") (See, e.g. Behr et al., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipidwith cholesterol ("DC-Chol") which has been formulated into liposomes incombination with DOPE. (See, Gao, X. and Huang, L., Biochim. Biophys.Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugatingpolylysine to DOPE, has been reported to be effective for transfectionin the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta1065:8, 1991). For certain cell lines, these liposomes containingconjugated cationic lipids, are said to exhibit lower toxicity andprovide more efficient transfection than the DOTMA-containingcompositions.

However, of the cationic lipids which have been proposed for use indelivering agents to cells, no particular cationic lipid has beenreported to work well with a wide variety of cell types. Since celltypes differ from one another in membrane composition, differentcationic lipid compositions and different types of lipid aggregates maybe effective for different cell types, either due to their ability tocontact and fuse with particular target cell membranes directly or dueto different interactions with intracellular membranes or theintracellular environment.

Thus, there remains a need for improved cationic lipids which arecapable of delivering macromolecules to a wide variety cell types withgreater efficiency.

SUMMARY OF THE INVENTION

The present invention provides compositions of novel dimeric cationiclipids, conjugates of these lipids with other molecules, and aggregatesof these cationic lipids with polyanionic macromolecules. The inventionfurther provides methods for their synthesis, methods of use and a kitfor delivering polyanionic macromolecules using these novel cationiclipids.

In one aspect of the present invention provided are dimeric cationiclipids having the structure:

    Z-S-S-Z[X.sup.- ].sub.m

wherein Z is ##STR1## wherein (a) n is 0, 1 or 2;

(b) R₁ is hydroxy, a glyceryl moiety or a lipophilic moiety;

(c) R₂ is

(i) --NH--[alk₁ --NH]_(n1) -- wherein n1 is an integer from 0 to 2 andalk₁ is an alkylene group of 2 to 6 carbon atoms;

(ii) -[W₁ ]_(n2) - wherein n2 is an integer from 0 to 3 and each W₁ isan independently selected amino acid residue;

(iii) --N(R₄)(alk₂)- wherein R₄ is hydrogen, alkyl of 1 to 18 carbonatoms optionally mono-, di- or tri-substituted with Y₁, Y₂ and/or Y₃ ;alkenyl of 2 to about 12 carbon atoms, aryl of about 6 to about 14carbon atoms and aralkyl of about 7 to about 15 carbon atoms and alk₂ isa straight chained or branched chain alkylene group of 1 to 18 carbonatoms optionally mono-, di- or tri-substituted with Y₁, Y₂ and/or Y₃ ;where Y₁, Y₂ and Y₃ are independently selected from the group consistingof arylamine of 5 to about 10 carbon atoms, aralkylamine of 5 to about10 carbon atoms, heterocylic amine, fluorine, a guanidinium moiety, anamidinium moiety, --NH₂, --NHR₁₀, --NR₁₀ R₁₁ and --N(R₁₀ R₁₁ R₁₂)wherein R₁₀, R₁₁ and R₁₂ are as defined hereinbelow;

(d) R₃ is

(i) --NH--[alk₃ ]_(n3) -H wherein n3 is an integer from 0 to 4 and alk₃is an alkylene group of 2 to 6 carbon atoms;

(ii) -[W₂ ]_(n4) H wherein n4 is an integer from 0 to 3 and each W₂ isan independently selected amino acid residue;

(iii) a negatively charged group selected from the group consisting of-alk₄ C(O)O⁻ ; -alk₄ -S(O₂)O⁻ ; -alk₄ P(O) (O⁻)O⁻ and -alk₄ OP(O) (O⁻)(O⁻) wherein alk₄ is an alkylene group of 1 to 6 carbon atoms;

(iv) heterocyclo of 4 to about 10 ring atoms with the ring atomsselected from carbon and heteroatoms, wherein the heteroatoms areselected from the group consisting of oxygen, nitrogen and S(O)_(i)wherein i is 0, 1 or 2;

(v) alkyl of 1 to about 12 carbon atoms optionally substituted with asubstituent selected from fluoro, a guanidinium moiety, an amidiniummoiety, --NH₂, --NHR₁₀, --NR₁₀ R₁₁ or NR₁₀ R₁₁ R₁₂ wherein each of R₁₀,R₁₁ and R₁₂ is independently selected from alkyl of 1 to about 12 carbonatoms, alkyl of 1 to about 12 carbon atoms substituted with 1 to about25 fluorine atoms and alkenyl of 2 to about 12 carbon atoms; or

(vi) W-(CH₂)_(t) --NH--(CH₂)_(q) -- wherein t and q are independentlyselected integers from 2 to 6 and W is a guanidinium moiety, anamidinium moiety, --NH₂, --NHR₁₀, --NR₁₀ R₁₁ or --NR₁₀ R₁₁ R₁₂ whereinR₁₀, R₁₁ and R₁₂ are as defined hereinabove

(e) X⁻ is an anion or a polyanion; and

(f) m is an integer selected such that [X⁻ ] is equal to the positivecharge of the lipid.

Also included within the scope of our invention is a lipid of the abovewithout the counter ion [X⁻ ]_(m)

According to one aspect of the invention, R₁ is a lipophilic moiety.Suitable lipophilic moieties include, but are not limited to, asymmetrical branched alkyl or alkenyl moiety of about 10 to about 50carbon atoms, a unsymmetrical branched alkyl or alkenyl moiety of about10 to about 50 carbon atoms, a steroidyl moiety (such as cholesteryl), aglyceryl moiety, the group --OCH(R₆ R₇) wherein R₆ and R₇ areindependently selected alkyl groups of about 10 to about 50 carbonatoms, --N(R₈ R₉), wherein R₈ and R₉ are independently selected alkyl oralkenyl groups of about 10 to about 50 carbon or taken together form acyclic amine group of about 4 to about 10 carbon atoms.

Where R₂ is --N(R₄) (alk₂ H) and R₄ is a substituted alkyl moiety of 1to about 18 carbon atoms, the substituted alkyl moiety may besubstituted with 1 to 3 substituents selected from an arylamine moietyof about 5 to about 10 carbon atoms, an aralkylamine of about 5 to about10 carbon atoms, a heterocyclic amine, F, a guanidinium moiety, anamidinium moiety, --NH₂, --NHR₁₀, --N(R₁₀ R₁₁), and --N(R₁₀ R₁₁ R₁₂)wherein R₁₀, R₁₁, and R₁₂ are as defined herein above.

Preferred R₂ groups include --NH[alk,NH]_(n1) --H wherein n1 is 0 or 1.Preferred alk, groups include --(CH₂)₃ --.

When R₂ is -[W₁ ]_(n1), suitable W₁ groups include amino acid residuesoptionally substituted with an alkyl of 1 to about 12 carbon atoms orwherein the amino group(s) is substituted to form a secondary, tertiary,or quaternary amine with an alkyl moiety of 1 to about 12 carbon atoms.Preferred amino acid residues include lysine, arginine, histidine,ornithine, tryptophane, phenylalanine, or tyrosine. Alternatively W₁ maybe an amino acid analog. Suitable amino acid analogs include3-carboxyspermidine, 5-carboxyspermidine, 6-carboxyspermine ormonoalkyl, dialkyl, or peralkyl substituted derivatives thereof whichare substituted on one or more amine nitrogens with a alkyl group of 1to about 12 carbon atoms.

Where R₂ is -[W₁ ]_(n2) H and n2 is 2 or 3, each W₁ may be independentlyselected, and R₂ may include natural amino acids, unnatural amino acidsor a combination of natural and unnatural amino acids.

When R₄ is a substituted alkyl group, suitable substitutions include thefollowing substituents -F, guanidinium moiety, amidinium moiety, --NH₂,--NHR₁₀, --N(R₁₀ R₁₁), and --N(R₁₀ R₁₁ R₁₂) wherein R₁₀, R₁₁ and R₁₂ asdefined herein above.

Suitable anions, X, include pharmaceutically acceptable anions andpolyanions. Preferred pharmaceutically acceptable anions and polyanionsinclude trifluoroacetates.

In addition, according to another aspect, the invention providescompositions comprising a anionic macromolecule and a lipid of thepresent invention. Suitable anionic macromolecules include an expressionvector capable of expressing a polypeptide in a cell and an Oligomer,more preferably DNA or RNA.

Particular preferred lipids of the present invention, include but arenot limited, to the following compounds: ##STR2##

In an alternate aspect, the present invention provides compositionswhich comprise a mixture of a lipid of the present invention mixed witha second lipid. Preferred are compositions which comprise a compound ofthe present invention and a second lipid such as those described in WO97/03939, and particularly preferred second lipids include Lipid A,Lipid B and Lipid C as depicted in FIG. 3. Preferred lipids of thepresent invention include Lipids 1-3, 1-1D and 2 as depicted in FIG. 2.The lipids may be present in a variety of ratios, preferably from about1:2 to about 20:1. Preferably the lipids are present in a 1:1weight:weight mixture. Particularly preferred is a mixture of 1-3 andLipid A.

The invention further provides methods for delivering a anionicmacromolecule into a cell by contacting said cell with a composition ofthe present invention. Also provided are methods for interfering withthe expression of a preselected in a cell by contacting said cell with acomposition of the present invention wherein the anionic macromoleculeis an Oligomer having a nucleoside base sequence which is substantiallycomplimentary to an RNA or DNA sequence in the cell that encodes thepreselected protein.

Definitions

In accordance with the present invention and as used herein, thefollowing terms are defined with the following meanings, unlessexplicitly otherwise.

The term "amino acid" refers to both natural and unnatural amino acidsin either their L- or D-forms. Natural amino acids include alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine andvaline. For example, unnatural amino acids include, but are not limitedto azetidinecarboxylic acid, 2-amincadipic acid, 3-aminoadipic acid,β-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyricacid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyricacid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4 diaminoisobutyricacid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid,N-ethylglycine, N-ethylasparagine, hydroxylysine, allohydroxylysine,3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine,N-methylglycine, N-methylisoleucine, N-methylvaline, norvaline,norleucine, ornithine and pipecolic acid.

The term "amino acid residue" refers to --NH--CH(R)--CO--, wherein R isthe side chain group distinguishing each amino acid. For cyclic aminoacids, the residue is ##STR3## wherein p is 1, 2 or 3 representing theazetidinecarboxylic acid, proline or pipecolic acid residues,respectively.

The term "alkyl" refers to saturated aliphatic groups includingstraight-chain, branched-chain and cyclic groups.

The term "alkenyl" refers to unsaturated hydrocarbyl group which containat lest one carbon-carbon double bond and includes straight-chain,branched-chain and cyclic groups.

The term "aryl" refers to aromatic groups which have at least one ringhaving a conjugated pi electron system and includes carbocyclic aryl,heterocyclic aryl and biaryl groups, all of which may be optionallysubstituted.

The term "aralkyl" refers to an alkyl group substituted with an arylgroup. Suitable aralkyl groups include benzyl, picolyl, and the like,all of which may be optionally substituted.

The term "aralkenyl" refers to an alkenyl group substituted with an arylgroup. Suitable aralkenyl groups include styrenyl and the like, all ofwhich may be optionally substituted.

The term "alkoxy" refers to the group --OR wherein R is alkyl.

The term "alkenyloxy" refers to the group --O--R wherein R is alkenyl.

The term "aryloxy" refers to the group --O--R wherein R is aryl.

The term "aralkyloxy" refers to the group --O--R wherein R is aralkyl.

The term "alkylene" refers to a divalent straight chain or branchedchain saturated aliphatic radical.

The term "alkylenecarboxy" refers to the group -alk-COOH where alk isalkylene.

The term "carboxamide" refers to the group --C(0)--NH₂.

The term "alkylenecarboxamide" refers to the group -alk-C(O)NH₂ wherealk is alkylene.

The term "alkylenehydroxy" refers to the group -alk-OH wherein alk isalkylene.

The term "methylene" refers to --CH₂ --.

The term "perfuoroalkyl" refers to an alkyl group wherein each hydrogenis replaced by a fluoro. Suitable perfluoroalkyl groups includeperfluoromethyl (having the structure of CF₃ --) and perfluroethyl(having the structure of CF₃ --CF₂ --) and the like.

The term "lipophilic moiety" refers to a moiety has one or more of thefollowing characteristics: is water insoluble, is soluble in non-polarsolvent, favors octanol in octanol/water partition measurements, or iscompatible with lipid bilayers and may be bilayer forming.

The term "aralkylamine" refers to an alkylamine substituted with an arylgroup. Suitable aralkyl groups include benzyl and other alkylsubstituted heterocycles and the like, all of which may be optionallysubstituted.

The term "aralkyl" refers to an alkyl group substituted with an arylgroup. Suitable aralkyl groups include benzyl and other alkylsubstituted heterocycles and the like, all of which may be optionallysubstituted.

The term "heterocyclic" refers to a group having from 1 to 4 heteroatomsas ring atoms in the aromatic ring and the remainder of the ring atomsare carbon atoms. Suitable heteroatoms include but are not limited tooxygen, nitrogen, sulfur, and selenium.

The term "heterocyclo" refers to a reduced heterocyclic ring systemcomprised of carbon, nitrogen, oxygen and/or sulfur atoms,and includesthose heterocyclic systems described in "Handbook of Chemistry andPhysics", 49th edition, 1968, R. C. Weast, editor; The Chemical RubberCo., Cleveland, Ohio. See particularly Section C, Rules for NamingOrganic Compounds, B. Fundamental Heterocyclic Systems.

The term "steroidyl" refers to a group of lipids that contain ahydrogenated cyclopentanoperhydrophenanthrene ring system. A preferedsteroidyl moiety is cholesteryl.

The term "glyceryl" refers to a mono-, di-, or trivalent radical formedby removal of a hydrogen from one, two, or three of the hydroxyl groupsof a glycerol molecule, which is a trihydric sugar alcohol of theformula CH₂ OHCHOHCH₂ OH.

The term "arylamine" refers to aromatic groups that have at least onering having a conjugated pi electron system and includes carbocyclicaryl, heterocyclic aryl, and biaryl groups, all of which are substitutedwith an amine.

The term "substantially complementary" refers to the Watson-Crick basepairing of the nucleosides of the target oligonucleotide sequence withthe nucleosides of the oligomer provided by this invention. It ispreferable that the sequence of the oligomer have sufficientcomplimentarity to bind and interfere with gene expression of the targetoligonucleotide. Preferably the oligomer is at least 50% complimentaryto the target oligonucleotide, more preferably at least 70% and mostpreferably at least 80%.

The term "amidinium" refers to the substituent of amidine which includesany compound having the monovalent group --C(NH)(NH₂).

The term "oligonucleoside" or "Oligomer" refers to a chain ofnucleosides that are linked by internucleoside linkages that isgenerally from about 4 to about 100 nucleosides in length, but which maybe greater than about 100 nucleosides in length. They are usuallysynthesized from nucleoside monomers, but may also be obtained byenzymatic means. Thus, the term "oligomer" refers to a chain ofoligonucleosides that have internucleosidyl linkages linking thenucleoside monomers and, thus, includes oligonucleotides, nonionicoligonucleoside alkyl- and aryl-phosphonate analogs, alkyl- andaryl-phosphonothioates, phosphorothioate or phosphorodithioate analogsof oligonucleotides, phosphoramidate analogs of oligonucleotides,neutral phosphate ester oligonucleoside analogs, such asphosphotriesters and other oligonucleoside analogs and modifiedoligonucleosides, and also includes nucleoside/non-nucleoside polymers.The term also includes nucleoside/non-nucleoside polymers wherein one ormore of the phosphorus group linkages between monomeric units has beenreplaced by a non-phosphorous linkage such as a formacetal linkage, athioformacetal linkage, a morpholino linkage, a sulfamate linkage, asilyl linkage, a carbamate linkage, an amide linkage, a guanidinelinkage, a nitroxide linkage or a substituted hydrazine linkage. It alsoincludes nucleoside/non-nucleoside polymers wherein both the sugar andthe phosphorous moiety have been replaced or modified such as morpholinobase analogs, or polyamide base analogs. It also includesnucleoside/non-nucleoside polymers wherein the base, the sugar, and thephosphate backbone of the non-nucleoside are either replaced by anon-nucleoside moiety or wherein a non-nucleoside moiety is insertedinto the nucleoside/non-nucleoside polymer. optionally, saidnon-nucleoside moiety may serve to link other small molecules which mayinteract with target sequences or alter uptake into target cells.

The term "polyfunctional linkers" refers to any polymer containingreactive atoms or reactive side chains that can be covalently linked tocationic lipid subunits as those described in the present invention, forexample polyfunctional linkers may include but are not limited topolyethylenamine, polypropylenamine, polybutylenamine, polyethyleneglycol, oxidized dextran, polyacrylamide, polylysine or a polypeptidederivative having reactive side chains. Such polypeptide derivativereactive side chains include for example the amino acid side chains oflysine, arginine, methionine, histidine, glutamine, asparagine,serine,threonine, glutamate and aspartate.

The term "lipid aggregate" includes liposomes of all types bothunilamellar and multilamellar as well as micelles and more amorphousaggregates of cationic lipid or lipid mixed with amphipathic lipids suchas phospholipids.

The term "target cell" refers to any cell to which a desired compound isdelivered, using a lipid aggregate as carrier for the desired compound.

The term "transfection" refers to the delivery of expressible nucleicacid to a target cell, such that the target cell is rendered capable ofexpressing said nucleic acid. It will be understood that the term"nucleic acid" includes both DNA and RNA without regard to molecularweight, and the term "expression" means any manifestation of thefunctional presence of the nucleic acid within the cell, includingwithout limitation, both transient expression and stable expression.

The term "delivery" refers to a process by which a desired compound istransferred to a target cell such that the desired compound isultimately located inside the target cell or in, or on the target cellmembrane. In many uses of the compounds of the invention, the desiredcompound is not readily taken up by the target cell and delivery vialipid aggregates is a means for getting the desired compound into thecell. In certain uses, especially under in vivo conditions, delivery toa specific target cell type is preferable and can be facilitated bycompounds of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the synthesis path of compound1-3 (See Examples 1-3).

FIG. 2 depicts structures of certain preferred lipids of the presentinvention.

FIG. 3 depicts the structures of Lipid A, Lipid B and Lipid C which maybe used in combination with the present. These lipids are described inWO 97/03939.

DETAILED DESCRIPTION OF THE INVENTION

All references cited below are hereby incorporated by reference in theirentirety.

The generic structure of functionally active cationic lipids requiresthree contiguous moieties, e.g. cationic-head-group, linker, andlipid-tail group. While a wide range of structures can be envisioned foreach of the three moieties, it has been demonstrated that there is no apriori means to predict which cationic lipid will successfully transfectanionic macromolecules into a particular cell line. The property of acationic lipid to be formulated with an anionic macromolecule which willthen successfully transfect a cell line is empirical. We demonstrate theabilities of novel cationic lipids which are chemically linked intomultimeric constructions to enhance the uptake of macromolecules.

The novel dimeric cationic lipids of the present invention have thegeneral structure:

    Z-S-S-Z[X.sup.- ].sub.m

wherein Z is ##STR4## wherein R₁, R₂, R₃ and n are as definedhereinabove.

R₁ represents the lipid-tail group of the dimeric cationic lipid and maybe hydroxyl, a glyceryl moiety, or a lipophilic moiety. In particular,suitable lipophilic moieties include, for example, a symmetricalbranched alkyl or alkenyl of about 10 to about 50 carbon atoms, aunsymmetrical branched alkyl or alkenyl of about 10 to about 50 carbonatoms, a amine derivative, a steroidyl moiety, --OCH(R₆ R₇), --NH(R₈) or--N(R₈ R₉), wherein R₈ and R₉ are independently an alkyl or alkenylmoiety of about 10 to about 50 carbon atoms, or together form a cyclicamine moiety of about 4 to about 10 carbon atoms. Preferred R₈ and R₉groups include --C₁₆ H₃₇.

In the case where R₁ is a steriodal moiety, suitable moieties include,for example, pregnenolone, progesterone, cortisol, corticosterone,aldosterone, androstenedione, testosterone, and cholesterol or analogsthereof.

The counterion represented by X⁻ is an anion or a polyanion that bindsto the positively charged groups present on the dimeric cationic lipidvia charge-charge interactions. When these cationic lipids are to beused in vivo the anion or polyanion should be pharmaceuticallyacceptable.

m is an integer indicating the number of anions or polyanions associatedwith the cationic lipid. In particular this integer ranges in magnitudefrom 0 to a number equivalent to the positive charge(s) present on thelipid.

n is an integer indicating the number of repeating units enclosed by thebrackets. Preferably n is an integer from 0 to 2.

The cationic lipids of the present invention include enantiomericisomers resulting from any or all asymmetric atoms present in the lipid.Included in the scope of the invention are racemic mixtures,diastereomeric mixtures, optical isomers or synthetic optical isomerswhich are isolated or substantially free of their enantiomeric ordiasteriomeric partners. The racemic mixtures may be separated intotheir individual, substantially optically pure isomers by techniquesknown in the art, such as, for example, the separation of diastereomericsalts formed with optically active acid or base adjuncts followed byconversion back to the optically active substances. In most instances,the desired optical isomer is synthesized by means of stereospecificreactions, beginning with the appropriate stereoisomer of the desiredstarting material. Methods and theories used to obtain enriched andresolved isomers have been described (Jacques et al., "Enantiomers,Racemates and Resolutions." Kreiger, Malabar, Fla., 1991).

Exemplary cationic lipids of the invention have the structures shown inthe Summary of the Invention above.

Preferred Compositions and Formation of Lipid Aqgregrates

The cationic lipids form aggregates with anionic or polyanionicmacromolecules such as oligonucleotides, oligomers, peptides, orpolypeptides through attraction between the positively charged lipid andthe negatively charged anionic macromolecule. The aggregates maycomprise multilamellar or unilamellar liposomes or other particles.Hydrophobic interactions between the cationic lipids and the hydrophobicsubstituents in the anionic or polyanionic macromolecule such asaromatic and alkyl moieties may also facilitate aggregate formation.Cationic lipids have been shown to efficiently deliver nucleic acids andpeptides into cells and thus are suitable for use in vivo or ex vivo.

Cationic lipid-anionic macromolecule aggregates may be formed by avariety of methods known in the art. Representative methods aredisclosed by Felgner et al., supra; Eppstein et al. supra; Behr et al.supra; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al.Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci.75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984;Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al.Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipidaggregates of appropriate size for use as delivery vehicles includesonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al.Biochim. Biophys. Acta 858:161, 1986). Microfluidization is used whenconsistently small (50 to 200 nm) and relatively uniform aggregates aredesired (Mayhew, supra). In general, aggregates may be formed bypreparing lipid particles consisting of either (1) a cationic lipid ofthe invention or (2) a cationic lipid mixed with a colipid, followed byadding a anionic macromolecule to the lipid particles at about roomtemperature (about 18 to 26° C.). In general, conditions are chosen thatare not conducive to deprotection of protected groups. The mixture isthen allowed to form an aggregate over a period of about 10 minutes toabout 20 hours, with about 15 to 60 minutes most conveniently used. Thecomplexes may be formed over a longer period, but additional enhancementof transfection efficiency will not usually be gained by a longer periodof complexing. Colipids may be natural or synthetic lipids having no netcharge or a positive or negative charge. In particluar, natural colipidsthat are suitable for preparing lipid aggregates with the cationiclipids of the present invention are dimyristoylphosphatidylethanolamine,dipalmitoyl-phosphatidylethanolamine,palmitoyloleolphosphatidyl-ethanolamine, cholesterol,distearoyalphosphatidyl-ethanolamine, phosphatidylethanolamine,phosphatidylethanolamine covalently linked to polyethylene glycol andmixtures of these colipids.

The optimal cationic lipid:colipid ratios for a given cationic lipid isdetermined by mixing experiments to prepare lipid mixtures foraggregation with a anionic macromolecule using cationic lipid:colipidratios between about 1:0.1 and 1:10. Methods to determine optimalcationic lipid:colipid ratios have been described (see, Felgner, infra).Each lipid mixture is optionally tested using more than oneoligonucleotide-lipid mixture having different nucleic acid:lipid molarratios to optimize the oligonucleotide:lipid ratio.

Suitable molar ratios of cationic lipid:colipid are about 0.1:1 to1:0.1, 0.2:1 to 1:0.2, 0.4:1 to 1:0.4, or 0.6:1 to 1:0.6. Lipid particlepreparation containing increasing molar proportions of colipid have beenfound to enhance oligonucleotide transfection into cells with increasingcolipid concentrations.

In addition, the cationic lipids can be used together in admixture, ordifferent concentrations of two or more cationic lipids in admixture,with or without colipid.

Liposomes or aggregates may be conveniently prepared by first drying thelipids in solvent (such as chloroform) under reduced pressure. Thelipids may then be hydrated and converted to liposomes or aggregates byadding water or low ionic strength buffer (usually less than about 200mM total ion concentration) followed by agitating (such as vortexingand/or sonication) and/or freeze/thaw treatments. The size of theaggregates or liposomes formed range from about 40 nm to 600 nm indiameter.

The amount of an oligonucleotide delivered to a representative cell byat least some of the cationic lipids was found to be significantlygreater than the amount delivered by commercially available transfectionlipids. The amount of oligonucleotide delivered into cells was estimatedto be about 2- to 100-fold greater for the cationic lipids of theinvention based on the observed fluorescence intensity of transfectedcells after transfection using a fluorescently labeled oligonucleotide.The cationic lipids described herein also transfect some cell types thatare not detectably transfected by commercial lipids. Functionality ofcationic lipid-DNA aggregates was demonstrated by assaying for the geneproduct of the exogenous DNA. Similarly, the functionality of cationiclipid-oligonucleotide aggregates were demonstrated by antisenseinhibition of a gene product.

The cationic lipids described herein also differed from commerciallyavailable lipids by efficiently delivering an oligonucleotide into cellsin tissue culture over a range of cell confluency from about 50-100%.Most commercially available lipids require cells that are at arelatively narrow confluency range for optimal transfection efficiency.For example, Lipofectin™ requires cells that are 70-80% confluent fortransfecting the highest proportion of cells in a population. Thecationic lipids described herein may be used to transfect cells that areabout 10-50% confluent, however, it is preferable to transfect at aconfluency of 60% to 100% for optimal efficiency. Confluency ranges of60-95% or 60-90% are thus convenient for transfection protocols withmost cell lines in tissue culture.

The cationic lipid aggregates were used to transfect cells in tissueculture and the RNA and the DNA encoded gene products were expressed inthe transfected cells.

The cationic lipid aggregates may be formed with a variety ofmacromolecules such as oligonucleotides and oligomers. Oligonucleotidesused in aggregate formation may be single stranded or double strandedDNA or RNA, oligonucleotide analogs, or plasmids.

Preferred Anionic Macromolecules

In general, relatively large oligonucleotides such as plasmids or mRNAswill carry one or more genes that are to be expressed in a transfectedcell, while comparatively small oligonucleotides will comprise (1) abase sequence that is complementary (via Watson Crick or Hoogsteenbinding) to a DNA or RNA sequence present in the cell or (2) a basesequence that permits oligonucleotide binding to a molecule inside acell such as a peptide, protein, or glycoprotein. Exemplary RNAs includeribozymes and antisense RNA sequences that are complementary to a targetRNA sequence in a cell.

An oligonucleotide may be a single stranded unmodified DNA or RNAcomprising (a) the purine or pyrimidine bases guanine, adenine,cytosine, thymine and/or uracil: (b) ribose or deoxyribose; and (c) aphosphodiester group that linkage adjacent nucleoside moieties.Oligonucleotides typically comprise 2 to about 100 linked nucleosides.Typical oligonucleotides range in size from 2-10, 2-15, 2-20, 2-25,2-30, 2-50, 8-20, 8-30 or 2-100 linked nucleotides. Oligonucleotides areusually linear with uniform polarity and, when regions of invertedpolarity are present, such regions comprise no more than one polarityinversion per 10 nucleotides. One inversion per 20 nucleotides istypical. Oligonucleotides can also be circular, branched ordouble-stranded. Antisense oligonucleotides generally will comprise asequence of about from 8-30 bases or about 8-50 bases that issubstantially complementary to a DNA or RNA base sequence present in thecell. The size of oligonucleotide that is delivered into a cell islimited only by the size of anionic macromolecules that can reasonablybe prepared and thus DNA or RNA that is 0.1 to 1 Kilobase (Kb), 1 to 20Kb, 20 Kb to 40 Kb or 40 Kb to 1,000 Kb in length may be delivered intocells.

Oligonucleotides also include DNA or RNA comprising one or more covalentmodifications. Covalent modifications include (a) substitution of anoxygen atom in the phosphodiester linkage of an polynucleotide with asulfur atom, a methyl group or the like, (b) replacement of thephosphodiester group with a nonphosphorus moiety such as --O--CH₂ O--,--S--CH₂ O-- or --O--CH₂ O--S, and (c) replacement of the phosphodiestergroup with a phosphate analog such as --O--P(S)(O)--O,--O--P(S)(S)--O--, --O--P(CH₃)(O)--O or --O--P(NHR₁₀)(O)--O-- where R₁₀is alkyl of 1 to about 6 carbon atoms, or an alkyl ether of 1 to about 6carbon atoms. Such substitutions may constitute from about 10% to 100%or about 20% to about 80% of the phosphodiester groups in unmodified DNAor RNA. Other modifications include substitutions of or on sugar moietysuch as morpholino, arabinose 2'-fluororibose, 2'-fluoroarabinose,2'-O-methylribose, or 2'-O-allylribose. Oligonucleotides and methods tosynthesize them have been described (for example see: PCT/US90/03138,PCT/US90/06128, PCT/US90/06090, PCT/US90/06110, PCT/US92/03385,PCT/US91/08811, PCT/US91/03680, PCT/US91/06855, PCT/US91/01141,PCT/US92/10115, PCT/US92/10793, PCT/US93/05110, PCT/US93/05202,PCT/US92/04294, WO 86/05518, WO 89/12060, WO 91/08213, WO 90/15065, WO91/15500, WO 92/02258, WO 92/20702, WO 92/20822, WO 92/20823, U.S. Pat.No. 5,214,136 and Uhlmann Chem. Rev. 90:543, 1990).

The linkage between the nucleotides of the oligonucleotide may be avariety of moieties including both phosphorus-containing moieties andnon phosphorus-containing moieties such as formacetal, thioformacetal,riboacetal and the like. A linkage usually comprises 2 or 3 atomsbetween the 5' position of a nucleotide and the 2' or 3' position of anadjacent nucleotide. However, other synthetic linkers may containgreater than 3 atoms.

The bases contained in the oligonucleotide may be unmodified or modifiedor natural or unnatural purine or pyrimidine bases and may be in the αor β anomer form. Such bases may be selected to enhance the affinity ofoligonucleotide binding to its complementary sequence relative to basesfound in native DNA or RNA. However, it is preferable that modifiedbases are not incorporated into an oligonucleotide to an extent that itis unable to bind to complementary sequences to produce a detectablystable duplex or triplex.

Exemplary bases include adenine, cytosine, guanine, hypoxanthine,inosine, thymine, uracil, xanthine, 2-aminopurine, 2,6-diaminopurine,5-(4-methylthiazol-2-yl)uracil, 5-(5-methylthiazol-2-yl)uracil,5-(4-methylthiazol-2-yl)cytosine, 5-(5-methylthiazol-2-yl)cytosine andthe like. Other exemplary bases include alkylated or alkynylated baseshaving substitutions at, for example, the 5 position of pyrimidines thatresults in a pyrimidine base other than uracil, thymine or cytosine,(i.e., 5-methylcytosine, 5-(1-propynyl)cytosine, 5-(1-butynyl)cytosine,5-(1-butynyl)uracil, 5-(1-propynyl)uracil and the like). The use ofmodified bases or base analogs in oligonucleotides have been previouslydescribed (see PCT/US92/10115; PCT/US91/08811; PCT/US92/09195; WO92/09705; WO 92/02258; Nikiforov, et al., Tet. Lett. 33:2379, 1992;Clivio, et al., Tet. Lett. 33:65, 1992; Nikiforov, et al., Tet. Lett.32:2505, 1991; Xu, et al., Tet. Lett. 32:2817, 1991; Clivio, et al.,Tet. Lett. 33:69, 1992; and Connolly, et al., Nucl. Acids Res. 17:4957,1989).

Use of Compositions and Lipid Aggregates

Aggregates may comprise oligonucleotides or oligomers encoding atherapeutic or diagnostic polypeptide. Examples of such polypeptidesinclude histocompatibility antigens, cell adhesion molecules, cytokines,antibodies, antibody fragments, cell receptor subunits, cell receptors,intracellular enzymes and extracellular enzymes or a fragment of any ofthese. The oligonucleotides also may optionally comprise expressioncontrol sequences and generally will comprise a transcriptional unitcomprising a transcriptional promoter, an enhancer, a transcriptionalterminator, an operator or other expression control sequences.

Oligonucleotides used to form aggregates for transfecting a cell may bepresent as more than one expression vector. Thus, 1, 2, 3, or moredifferent expression vectors may be delivered into a cell as desired.Expression vectors will typically express 1, 2, or 3 genes whentransfected into a cell, although many genes may be present such as whena herpes virus vector or a artificial yeast chromosome is delivered intoa cell. Expression vectors may further encode selectable markers (e.g.neomycin phosphotransferase, thymidine kinase, xanthine-guaninephosphoribosyl-transferase, and the like) or biologically activeproteins such as metabolic enzymes or functional proteins (e.g.immunoglobulin genes, cell receptor genes, cytokines (e.g. IL-2, IL-4,GM-CSF, γ-INF, and the like)), or genes that encode enzymes that mediatepurine or pyrimidine metabolism and the like.

The nucleic acid sequence of the oligonulcleotide coding for specificgenes of interest may be retrieved, without undue experimentation, fromthe GenBank of EMBL DNA libraries. Such sequences may include codingsequences, for example, the coding sequences for structural proteins,hormones, receptors and the like, and the DNA sequences for other DNAsof interest, for example, transcriptional and translational regulatoryelements (promoters, enhancers, terminators, signal sequences and thelike), vectors (integrating or autonomous), and the like. Non-limitingexamples of DNA sequences which may be introduced into cells includethose sequences coding for fibroblast growth factor (see WO 87/01728);ciliary neurotrophic factor (Lin et al., Science, 246:1023, 1989); humaninterferon-α receptor (Uze, et al., Cell, 60:225, 1990); theinterleukins and their receptors (reviewed in Mizal, FASEB J., 3:2379,1989); hybrid interferons (see EPO 051,873); the RNA genome of humanrhinovirus (Callahan, Proc. Natl. Acad. Sci., 82:732, 1985); antibodiesincluding chimeric antibodies (see U.S. Pat. No. 4,816,567); reversetranscriptase (see Moelling, et al., J. Virol., 32:370, 1979); human CD4and soluble forms thereof (Maddon et al., Cell, 47:333, 1986, WO88/01304 and WO 89/01940); and EPO 330,191, which discloses a rapidimmunoselection cloning method useful for the cloning of a large numberof desired proteins.

Aggregates can be used in antisense inhibition of gene expression in acell by delivering an antisense oligonucleotide into the cell (seeWagner, Science 260:1510, 1993 and WO 93/10820). Such oligonucleotideswill generally comprise a base sequence that is complementary to atarget RNA sequence that is expressed by the cell. However, theoligonucleotide may regulate intracellular gene expression by binding toan intracellular nucleic acid binding protein (see Clusel, Nuc. AcidsRes. 21:3405, 1993) or by binding to an intracellular protein ororganelle that is not known to bind to nucleic acids (see WO 92/14843).A cell that is blocked for expression of a specific gene(s) is usefulfor manufacturing and therapeutic applications. Exemplary manufacturinguses include inhibiting protease synthesis in a cell to increaseproduction of a protein for a therapeutic or diagnostic application(e.g., reduce target protein degradation caused by the protease).Exemplary therapeutic applications include inhibiting synthesis of cellsurface antigens to reduce rejection and/or to induce immunologictolerance of the cell either after it is implanted into a subject orwhen the cell is transfected in vivo (e.g. histocompatibility antigens,such as MHC class II genes, and the like).

Methods to introduce aggregates into cells in vitro and in vivo havebeen previously described (see U.S. Pat. Nos. 5,283,185 and 5,171,678;WO 94/00569; WO 93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550,1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human GeneTher. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J.11:417, 1992.

Entry of liposomes or aggregates into cells may be by endocytosis or byfusion of the liposome or aggregate with the cell membrane. When fusiontakes place, the liposomal membrane is integrated into the cell membraneand the aqueous contents of the liposome merge with the fluid in thecell.

Endocytosis of liposomes occurs in a limited class of cells; those thatare phagocytic, or able to ingest foreign particles. When phagocyticcells take up liposomes or aggregates, the cells move the spheres intosubcellular organelles known as lysosomes, where the liposomal membranesare thought to be degraded. From the lysosome, the liposomal lipidcomponents probably migrate outward to become part of cell's membranesand other liposomal components that resist lysosomal degradation (suchas modified oligonucleotides or oligomers) may enter the cytoplasm.

Lipid fusion involves the transfer of individual lipid molecules fromthe liposome or aggregate into the plasma membrane (and vice versa); theaqueous contents of the liposome may then enter the cell. For lipidexchange to take place, the liposomal lipid must have a particularchemistry in relation to the target cell. Once a liposomal lipid joinsthe cell membrane it can either remain in the membrane for a period oftime or be redistributed to a variety of intracellular membranes. Thecationic lipids of the present invention can be used to deliver anexpression vector into a cell for manufacturing or therapeutic use. Theexpression vectors can be used in gene therapy protocols to deliver atherapeutically useful protein to a cell or for delivering nucleic acidsencoding molecules that encode therapeutically useful proteins orproteins that can generate an immune response in a host for vaccine orother immunomodulatory purposes according to known methods (see U.S.Pat. Nos. 5,399,346 and 5,336,615, WO 94/21807 and WO 94/12629). Thevector-transformed cell can be used to produce commercially useful celllines, such as a cell line for producing therapeutic proteins or enzymes(e.g. erythropoietin, and the like), growth factors (e.g. human growthhormone, and the like) or other proteins. The aggregates may be utilizedto develop cell lines for gene therapy applications in humans or otherspecies including murine, feline, bovine, equine, ovine or non-humanprimate species. The aggregates may be used to deliver anionicmacromolecules into cells in tissue culture medium in vitro or in ananimal in vivo.

To assist in understanding the present invention, the following examplesare included which describe the results of a series of experiments. Thefollowing examples relating to this invention should not, of course, beconstrued as specifically limiting the invention. Variations of theinvention, now known or later developed, which would be within thepurview of one skilled in the art are considered to fall within thescope of the invention as described herein and hereinafter claimed.

EXAMPLES

General Methods

All reactions were run under a positive pressure of dry argon. Reactionsrequiring anhydrous conditions were performed in flame-dried glasswarewhich was cooled under argon. Tetrahydrofuran (THF, Aldrich Milwaukee,Wis.) was distilled from potassium/benzophenone ketyl immediately priorto use. Methylene chloride, pyridine, toluene, heptane, methanol, andethanol were obtained as anhydrous reagent (<0.005% water) or reagentgrade and were used without further purification. TLC was performed on0.2 mm E. Merck precoated silica gel 60 F₂₅₄ TLC plates (20×20 cmaluminum sheets, Fisher, Pittsburgh, Pa.). Flash chromatography wasperformed using E. Merck 230-400 mesh silica gel. All ¹ H, ¹³ C and ³¹ PNMR spectra were recorded on a 300 MHz Bruker ARX Spectrometer (Bruker,Boston, Mass.)and were obtained in CDCl₃ unless otherwise indicated.Mass spectra were provided by The Scripps Research Institute MassSpectrometry Facility of La Jolla, Calif. FAB mass spectra were obtainedon a FISONS VG ZAB-VSE double focusing mass spectrometer equipped with acesium ion gun (Fisions, Altrincham, UK). ESI mass spectra were obtainedon an API III PE Sciex triple-quadrupole mass spectrometer (Sciex,Toronto, Calif.).

Example 1 Synthesis of Bis [N.sup.α-Boc-N',N'-Dioctadecyl]-L-Cystinamide (1-1)

Approximately 3.4 mmol of Boc-L-cystine, 6.8 mmol of dioctadecylamine,and 6.8 mmol of N-hydroxybenzotriazole were added to dry dichloromethane(60 mL). Dicyclohexylcarbodiimide (6.8 mmol) was dissolved in drydichloromethane (15 mL) and added to the reaction mixture. The reactionproceeded at room temperature for 21 hours. The dicyclohexylurea wasremoved by filtration and the desired product was purified by columnchromatography on silica gel (heptane:ethyl acetate, 1:1) (1.67 g, 34%yield) (see Scheme 1). ¹ H NMR (300 MHz, CDCl₃, TMS=0) δ 5.29 (d, 2H,J=9.0), 4.89 (m, 2H), 3.50-2.85(m, 12H), 1.70-1.42 (m, 10H), 1.43 (s,18H), 1.26 (bs, 128H), 0.88 (t, 12H, J=6.9).

Example 2 Synthesis of N²,N⁵ -Bis[(1,1-Dimethylethoxy)Carbonyl]-N²,N⁵-Bis[3-[(1,1-Dimethylethoxy)Carbonyl]Aminopropyl]-L-Ornithine,N-Hydroxysuccinimydyl Ester (1-2)

A 100 ml round-bottomed reaction flask was charged with (2.08 g, 3.2mmol) of N²,N⁵ -Bis[(1,1-dimethylethoxy)carbonyl]-N²,N⁵-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine (Behr, J.P. Acc. Chem. Res. 26:274, 1993), dicyclohexylcarbodiimide (0.73 g, 3.5mmol), N-hydroxysuccinimide (0.41 g, 3.5 mmol), and methylene chloride(20 mL). The reaction mixture was stirred for 5 hours and then placed ina refrigerator (0 to 5° C.) overnight (15 hours). This mixture wasfiltered and washed with methylene chloride, and the filtrate wasconcentrated by rotary vaporization. The crude product was purified byflash chromatography on silica gel using 1:1 ethyl acetate:heptane toprovide 1.2 g (50% yield) of 1-2 as a white solid: ¹ H NMR δ 5.26 (br s,1H), 4.77 (br s, 1H), 4.28 (br s, 1H), 3.22-3.09 (m, 10H), 2.84 (s, 4H),2.05-1.61 (m, 8H), 1.48 and 1.46 and 1.44 (3 s, 36H); MS (ESI) m/z 744(MH⁺)

Example 3 Synthesis of Bis[N.sup.α,N.sup.δ-Di(3-Aminopropyl)-L-Ornithinyl-N',N'-Dioctadecyl]-L-CystinamideOctahydrotrifluoroacetate(1-3)

Approximately 1.67 g of Bis[N.sup.α-Boc-N',N'-dioctadecyl]-L-cystinamide was dissolved in a mixture oftrifluoroacetic acid and dichloromethane. After 20 minutes the solventswere removed, and the residue was coevaporated from 1,2-dichloroethaneand ether. The crude product was placed under vacuum overnight to removetrace amounts of solvents. A white solid was obtained (1.88 g). Thedeprotected amino lipid (0.067 mmol) was dissolved in drydichloromethane (1 mL), and approximately 0.134 mmol of compound 1-2 wasadded. Approximately 0.67 mmol of Hunig's base was added, and thereaction proceeded at room temperature for 18 hours. After removing theHunig's base (salt) the product was purified by column chromatography onsilica gel (heptane:ethyl acetate, 1:1; R_(f) =0.37). The purifiedproduct was then deprotected with trifluoroacetic acid:dichloromethane1:1 (6 mL) for 20 minutes. After coevaporation from 1,2-dichloroethanethe desired product was obtained (120 mg, 75% yield). ¹ H NMR (300 MHz,CDCl₃ +CD₃ 0D, TMS=0) δ 5.12(m, 2H), 4.02(m, 2H) 3.55-3.30(m, 4H),3.25-2.85 (m, 24H), 2.25-1.40 (m, 20H), 1.27 (bs, 115H), 0.89 (t, 12H,J=6.9); ESI MS m/z calculated for C₁₀₀ H₂₀₆ N₁₂ O₄ S₂ : 1705, found 1706(M+H)⁺.

Example 4 Synthesis of Bis(L-ornithinyl)-N',N'-dioctadecyl-L-cystinamidetetrahydrotrifluoroacetate (2)

The title compound was prepared using an identical procedure asdescribed in Example 3 for the preparation of 1-3, with the exceptionthat di-Boc ornithine succinimidyl ester was used in place of reagent1-2.

Example A Preparation and transfection protocols for COS-7, SNB-19 RD,and C8161 cells with mixtures of cationic lipids and CAT plasmid

i. Culturing and Transfection of Cells

Cell lines were plated at 1.5×10⁵ cells/well in a 12 well plate formaton the day before transfection. Cultures were maintained at 37° C. in 5%CO₂. On the next day, when the cells reached approximately 80%confluence, the transfection mixes were prepared as follows: 126 μg ofthe target CAT plasmid pG1035 (described below) was added to 36.0 mL ofOpti-MEMO (Gibco/BRL, Gaithersburg, Md.) to make a plasmid stocksolution. 63 μg of each lipid mix (from a high concentration stock in100% ethanol) was added to individual 1.5 mL aliquots Opti-MEMO andmixed thoroughly. Then, 2 mL of the DNA stock (containing 7 μg ofplasmid) were added to each 1.5 mL aliquot of lipid/Opti-MEM® and gentlyvortexed. This procedure yielded 3.5 mL of plasmid/lipid mixture at 2μg/mL plasmid and 18 μg/mL lipid for a 9 to 1 lipid to DNA ratio. Thequantity of ethanol in the final cell cultures was 2% or less. Thissmall quantity of ethanol was confirmed to have no adverse effect on anyof the cell lines.

In order to prepare cells for transfection, the culture medium wasaspirated from the wells and the cells were rinsed twice in 1 mLOpti-MEM® per well. The transfection experiments were performed intriplicate; thus, 1 mL of each transfection mix was then added to eachof three wells. The cells were cultured in the transfection mix for 5 to6 hours. The transfection mix was then removed and replaced with 1 mL ofcomplete culture medium (DMEM or DMEM/F12 plus 10% fetal bovine serumand 1/100 dilution of penicillin/streptomycin stock, all from Gibco/BRL,(Gaithersburg, Md.) and the cells were allowed to recover overnightbefore expression of the CAT gene was measured.

Cell lysates were prepared by rinsing twice in PBS and then were treatedwith 0.5 mL of 1× Reporter Lysis Buffer (Promega, Madison, Wis.). Thelysed cells were pipetted into 1.5 mL tubes and frozen in CO₂ /EtOH onceand thawed. The crude lysate was then clarified by microcentrifugationat 14,000 rpm for 10 minutes to pellet cell debris. The clearsupernatant was recovered and assayed directly or stored at -20° C. forassay later.

The cell lysates were then assayed for CAT activity and the totalprotein concentration was determined as described below. The CATactivity was normalized to total protein and plotted as shown.

ii. Chloramphenicol Acetyltransferase Assay

This assay was performed generally as follows. First, the followingreaction mixture was prepared for each sample:

65 mL 0.23 M Tris, pH 8/0.5% BSA (Sigma, St. Louis, Mo.),

4 mL ¹⁴ C-chloramphenicol, 50 nCi/mL (Dupont, Boston, Mass.), and

5 mL mg/mL n-butyryl coenzyme A (Pharmacia, Piscataway, N.J.).

A CAT activity standard curve was prepared by serially diluting CATstock (Promega, Madison, Wis.) 1:1000, 1:10,000 and 1:90,000 in 0.25 MTris, pH 8/0.5% BSA. The original stock CAT was at 7000 Units/mL. CATlysate was then added in a labeled tube with Tris/BSA buffer to a finalvolume of 50 mL.

Approximately 74 mL of reaction mixture was then added to each sampletube, which was then typically incubated for approximately 1 hour in a37° C. oven. The reaction was terminated by adding 500 mL pristane:mixedxylenes (2:1) (Sigma, St. Louis, Mo.) to each tube. The tubes were thenvortexed for 2 minutes and spun for 5 minutes. Approximately 400 mL ofthe upper phase was transferred to a scintillation vial with 5 mLScintiverse (Fisher, Pittsburgh, Pa.). The sample was then counted in ascintillation counter (Packard).

iii. Coomassie Protein Assay

The total protein content of the clarified cell lysates was determinedby mixing 6 μL of each cell lysate to 300 mL of Coomassie protein assayreagent (Pierce, Rockford, Md.) in the wells of an untreated microtiterassay plate. Concentration curve standards were prepared using 6 μL of0, 75, 100, 200, 250, 400, 500, 1000, and 1500 mg/mL BSA stock solutionsand 300 mL of the Coomassie reagent. The assay samples were allowed tosit for approximately 30 minutes before reading the optical absorbanceat 570 nm in a microplate reader (Molecular Probes).

iv. Results

The cells were assayed for CAT protein as described above. Results ofthe transfection efficiency of the cationic lipids are set forth inTables 2 and 4.

Table 2 depicts demonstration of plasmid delivery and expression inCOS-7, SNB-19, RD and C-8161 cells. Table 4 depicts demonstration ofplasmid delivery in SBN-19 cells.

Example B FITC-Oligonucleotide Uptake Assay

i. Oligomers Used

The oligonucleotides used for the determination of cationic lipidmediated oligonucleotide uptake in all cell lines tested were:

    #3498-PS: 5'FITC-ggt-ata-tcc-agt-gat-ctt-ctt-ctc           [SEQ. ID NO. 1],

Oligomer 3498-PS has an all-phosphorothioate backbone. Thisoligonucleotide has 23 negative charges on the backbone and isconsidered to be 100% negatively charged.

    #3498: 5'FITC-ggt-ata-tcc-aqt-gat-ctt-ctt-ctc              [SEQ. ID NO. 2],

Oligomer 3498 is a chimeric oligonucleoside. The underlined bases werelinked by a phosphorothioate backbone, while the other linkages in theoligomer consisted of alternating methylphosphonates andphosphodiesters. The oligomer had 11 methylphonate, 7 diester, and 5phosphorothioates linkages. The total charge density was 57% of 3498-PS.

    #3793-2: 5'FITC-ggu-aua-ucc-agu-gau-cuu-cut                [SEQ. ID NO. 3],

Oligomer 3293-2 has an alternating methylphosphonate and diesterbackbone with all 2'-O-methyl groups on each ribose in theoligonucleotide. The total charge density was 50% of 3498-PS.

Stocks of oligomers 3498-PS and 3498 were prepared at 300 micromolar,while the oligomer 3793-2 stock was prepared at 440 micromolar.

ii. Reagents and Cells

The commercially available lipids used in the assays were:

Lipofectin® ("LFN") Lot#EF3101 1 mg/mL, Gibco/BRL (Gaithersburg, Md.)

LipofectAMINE® ("LFA") Lot#EFN101 2 mg/mL, Gibco/BRL (Gaithersburg, Md.)

Transfectam® ("TFM") Lot#437121 1 mg dry, Promega, (Madison, Wis.) andresuspended in 100% ethanol.

Tfx™--50 Reagent (Polygum), Catalog #E1811, Promega (Madison, Wis.).

CellFECTIN™ Reagent, Catalog #L0362-010, Life Technologies Gibco/BRL(Gaithersburg, Md.).

The novel lipids of the present invention used in these evaluations, aslisted in the data tables (Tables 1 through 4), were at 1 mg/mL in 100%ethanol.

The tissue culture cell stocks, SNB-19 (human glioblastoma), C8161 (ahuman amelanotic melanoma), LOX-IMVI (a human amelanotic melanoma), RD(human rhabdomyosarcoma, ATCC# CCL-136) and COS-7 (African green monkeykidney cells, ATCC # CRL-1651) were maintained in standard cell culturemedia: DMEM:F12 (1:1) mix from Mediatech, Lot#150901126, 10% fetalbovine serum from Gemini Bioproducts, Lot#A1089K, 100 units/mLpenicillin and 100 micrograms/mL streptomycin, from Mediatech,Lot#30001044 and 365 micrograms/mL L-glutamine. The cells weremaintained under standard conditions (37° C., 5% CO₂ atmosphere) at alltimes prior to fixation and microscopic examination.

LOX cells were obtained from Southern Research Institute, Cell Biologyand Immunology Group, 2000 Ninth Avenue South, Birmingham, Ala. 35205.SNB-19 cells were obtained from Richard Morrison, Ph.D., AssociateProfessor, University of Washington, School of Medicine, 1959 N.E.Pacific Street, Seattle, Wash. 98195. C8161 cells were obtained fromWilliam G. Cance, M.D., Professor, University of North Carolina, 3010Old Clinic Building, CB 7210, Chapel Hill, N.C. 27599.

iii. Preparation of Cells and Transfection Mixes

For each FITC labeled oligomer delivery determination, the appropriatecells were plated into 16 well slides (Nunc #178599, glass microscopeslide with 16 removable plastic wells attached to the slide surface witha silicone gasket) according to standard tissue culture methods. Eachcell line was plated at a starting density (approximately 20,000cells/well) that allowed them to be healthy and 60-80% confluent one totwo days after plating. The cells to were allowed to adhere to the glassand recover from the plating procedure in normal growth medium for 24 to48 hours before beginning the transfection procedure.

Oligonucleotide transfection mixes were made up in Opti-MEM® withoutantibiotics as follows: 500 μL aliquots of Opti-MEM® containing a 0.25micromolar solution of either oligomer 3498-PS, 3498, or 3793-2 (2micrograms of oligomer per sample) were pipetted into 1.5 mL Eppendorftubes. Cationic lipid or lipid mixture was then added to the oligomersolution to give a final 9:1 or 6:1 ratio (18 or 12 μg of lipid total)of cationic lipid to oligomer by weight, as listed in Tables 1 and 3A to3C. The tubes were mixed by vortexing immediately after the addition oflipid.

Prior to beginning the transfection reactions the cells were rinsed in200 μL Opti-MEM®; then, the cells were rinsed with Dulbecco's phosphatebuffered saline (PBS) solution, and 200 μL of oligomer transfection mixwas added directly to a well to begin each transfection reaction.Transfection reactions were allowed to continue for four to six hours.

At that time, the cells were then rinsed in PBS from Mediatech and fixedfor ten minutes in 200 μL of 3.7% formaldehyde (Sigma, St. Louis, Mo.)to terminate the transfection reaction. Then the wells were rinsed againin PBS. The formaldehyde was quenched with 200 μL of 50 mM glycine(Sigma, St. Louis, Mo.) for ten minutes. Finally, the wells were thenemptied by shaking out the glycine solution. At that time, the plasticchambers and silicone gasket were removed and the cells were coveredwith Fluoromount-G mounting medium (from Fisher, Pittsburgh, Pa., withphotobleaching inhibitors) and a cover slip.

Intracellular fluorescence was evaluated under 200× magnification with aNikon Labophot-2 microscope with an episcopic-fluorescence attachment.Using this equipment we could distinguish extracellular from nuclear andendosomal fluorescence.

The cells were scored for uptake of FITC labelled oligomer as follows:No nuclear fluorescence, 0; up to 20% fluorescent nuclei, 1; up to 40%fluorescent nuclei, 2; up to 60% fluorescent nuclei, 3; up to 80%fluorescent nuclei, 4; and up to 100% fluorescent nuclei, 5.

The results of the transfections in COS-7, SNB-19, C-8161, LOX and RDcells are tabulated in Tables 1 and 3A to 3C. Table 1 depictsdemonstration of nuclear delivery of three different oligonucleotideconstructs to COS-7, SNB-19, C-8161 and RD cells. Tables 3A to 3C depictdemonstration of nuclear delivery of three different oligonucleotideconstructs to COS-7 (Table 3A), SNB-19 (Table 3B and LOX (Table 3C)cells.

                                      TABLE 1                                     __________________________________________________________________________            Phosphorothioate                                                                             Chimera       Steric blocker                           Lipids  COS-7                                                                             SNB-019                                                                            C-8161                                                                            RD                                                                              COS-7                                                                             SCB-19                                                                            C-8161                                                                            RD                                                                              COS-7                                                                             SNB-19                                                                            C-8161                                                                            RD                           __________________________________________________________________________    Commercial                                                                      LipofectAMINE 4 3 4 4 5 4 5 5 5 4 5 5                                         Lipofectin 5 3 3 4 1 2 3 3 0 0 2 0                                            Genta                                                                         1-3 4 3 5 5 5 5 5 5 4 5 5 4                                                   Lipid A/1-3 4 3 4 4 5 5 5 5 3 5 5 5                                           Lipid A 2 3 3 3 2 3 4 4 1 3 3 4                                             __________________________________________________________________________

                  TABLE 2                                                         ______________________________________                                        Cell line Lipid       CAT CPM, mean                                                                             SDV                                         ______________________________________                                        COS-7     None        894         23                                             Transfectam 89437 14746                                                       1-3 71628 6351                                                                Lipid A/2 40338 4513                                                          Lipid A/1-3 91912 12908                                                      SNB-19 None 807 24                                                             Transfectam 106060 17596                                                      1-3 113466 20594                                                              Lipid A/2 117722 13766                                                        Lipid A/1-3 142476 12916                                                     RD None 743 32                                                                 Transfectam 51255 1490                                                        Lipid 1-3 35947 2247                                                          Lipid A/2 52019 5367                                                          Lipid A/1-3 103993 14323                                                     C-8161 None 851 32                                                             Transfectam 141138 2049                                                       1-3 153442 25861                                                              Lipid A/2 35180 2050                                                          Lipid A/1-3 150831 11361                                                   ______________________________________                                    

                                      TABLE 3A                                    __________________________________________________________________________           Delivery                                                                           Comments                                                                           T'fection                                                                          Delivery                                                                             Comments                                                                           T'fection                                                                          Delivery                                                                           Comments                                                                           T'Fection                      COS-7 Cells 3498 3498 Time 3498PS 3498PS Time 3793-2 3793-2 Time            __________________________________________________________________________    Commercial Lipids                                                             Celfectin                                                                            2    unusual                                                                            5 hr 4*          5 hr                                            nuclei                                                                      Lipofectamine   4, 5*  5 hr 3*, 4*  5 hr 5*  5 hr                             Lipofectin   1, 1*  5 hr 4*, 5*  5 hr 0*  5 hr                                Transfectam 5  5 hr (9:1), .5 (6:1)  5 hr 5* some dim 5 hr                    Polygum 4  5 hr                                                             Genta Chemistry Lipids                                                        1-1D   0         5 hr                                                           1-1D/Lipid A   3.5  5 hr 0   5 hr                                             2 0  5 hr                                                                     2/Lipid A 4.5, 4*  5 hr  1, 4* some dim 5 hr 2* toxic? 5 hr                   1-3 2  5 hr                                                                   1-3  5*  5 hr 4*  5 hr 4*  5 hr                                               1-3/Lipid A 4.5, 5*  5 hr  3, 4*  5 hr 3*  5 hr                             __________________________________________________________________________     *= 9:1                                                                        lipid:oligo                                                              

                                      TABLE 3B                                    __________________________________________________________________________           Delivery                                                                           Comments                                                                            T'fection                                                                          Delivery                                                                           Comments                                                                           T'fection                                                                          Delivery                                                                            Comments                                                                           T'Fection                      SNB 19 Cells 3498 3498 Time 3498PS 3498PS Time 3793-2 3793-2 Time           __________________________________________________________________________    Commercial Lipids                                                             Transfectam                                                                          5    very bright                                                                         5 hr varies    5 hr 4, 5       5 hr                           Lipofectamine 4  5 hr 4* many, dim 5 hr 4, 5 better than 5 hr                         TFA                                                                   Lipofectin 1  5 hr 4*  5 hr 1 very dim 5 hr                                   Celfectin 3  5 hr 3*  5 hr  very dim 5 hr                                   Genta Chemistry Lipids                                                        1-1D/Lipid A                                                                           1.5      5 hr  0.5      5 hr                                           2 3 dim 5 hr 2* few cells 5 hr 2 dim 5 hr                                     2/Lipid A   3.5  5 hr 1   5 hr 3, 2 toxic? 5 hr                               2/Lipid B 3 bright 4.5 hr   3* toxic 5 hr                                     2/Lipid C 1, 2  4.5 hr   3* toxic 5 hr                                        1-3 5   6 hr, 4*, 5* dim  6 hr, 4, 5 ++199  5 hr                                 5 hr   5 hr                                                                1-3/Lipid A 4  5 hr 3* dim 5 hr 4 toxic? 5 hr                                 1-3/Lipid B 5  4.5 hr   5*  5 hr                                              1-3/Lipid C 5  5 hr 5*  5 hr                                                __________________________________________________________________________     *= 9:1                                                                        lipid:oligo                                                              

                                      TABLE 3C                                    __________________________________________________________________________          Delivery                                                                           Comments                                                                           T'fection                                                                          Delivery                                                                           Comments                                                                           T'fection                                        LOX Cells 3498 3498 Time 3498PS 3498PS Time                                 __________________________________________________________________________    Commercial Lipids                                                             Transfectam                                                                         3         5 hr                                                            Celfectin 0  5 hr                                                             Lipofectin 0  5 hr                                                          Genta Chemistry Lipids                                                        2/Lipid A                                                                           3         5 hr                                                            1-3 2  5 hr 0*  5 hr                                                          1-3/Lipid A 2  5 hr                                                         __________________________________________________________________________     *= 9:1                                                                        lipid:oligo                                                              

                  TABLE 4                                                         ______________________________________                                        Cell line Lipid       CAT cpm/ug ave.                                                                           SDV                                         ______________________________________                                        SNB-19    Lipofectin  742         72                                             1-3 829 43                                                                    1-3/Lipid A 665 1                                                          ______________________________________                                    

We claim:
 1. A compound of the formula

    Z-S-S-Z

wherein Z is ##STR5## wherein (a) n is 0, 1 or 2; (b) R₁ is hydroxy, aglyceryl moiety or a lipophilic moiety; (c) R₂ is(i) --NH-[alk₁--NH]_(n1) -- wherein n1 is an integer from 0 to 2 and alk₁, is analkylene group of 2 to 6 carbon atoms; (ii) --[W₁ ]_(n2) - wherein n₂ isan integer from 0 to 3 and each W₁ is an independently selected aminoacid residue; (iii) --N(R₄) (alk₂ -) wherein R₄ is hydrogen, alkyl of 1to 18 carbon atoms optionally mono-, di- or tri-substituted with Y₁, Y₂and/or Y₃ ; alkenyl of 2 to about 12 carbon atoms, aryl of about 6 to 14carbon atoms and aralkyl of about 7 to about 15 carbon atoms and alk₂ isa straight chained or branched chain alkylene group of 1 to 18 carbonatoms optionally mono-, di- or tri-substituted with Y₁, Y₂ and/or Y₃where Y₁, Y₂ and Y₃ are independently selected from the group consistingof arylamine of 5 to about 10 carbon atoms, aralkylamine of 5 to about10 carbon atoms, heterocylic amine, fluorine, a guanidinium moiety, anamidinium moiety, --NH₂, --NHR₁₀, --NR₁₀ R₁₁, and --N(R₁₀ R₁₁ R₁₂)wherein R₁₀, R₁₁ and R₁₂ are as defined hereinbelow; (d) R, is(i)--NH-[alk₃ ]_(n3) -H wherein n3 is an integer from 0 to 4 and alk₃ is analkylene group of 2 to 6 carbon atoms; (ii) --[W₂ ]_(n4) H wherein n4 isan integer from 0 to 3 and each W₂ is an independently selected aminoacid residue; (iii) a negatively charged group selected from the groupconsisting of -alk₄ C(O)O⁻ ; -alk₄ -S(O₂)O⁻ ; -alk₄ P(O)(O⁻)O⁻ and -alk₄OP(O)(O⁻)(O⁻) wherein alk₄ is an alkylene group of 1 to 6 carbon atoms;(iv) heterocyclo of 4 to about 10 ring atoms with the ring atomsselected from carbon and heteroatoms, wherein the heteroatoms areselected from the group consisting of oxygen, nitrogen and S(O)_(i)wherein i is 0, 1 or 2; (v) alkyl of 1 to about 12 carbon atomsoptionally substituted with a substituent selected from fluoro, aguanidinium moiety, an amidinium moiety, --NH₂, --NHR₁₀, --NR₁₀ R₁₁ orNR₁₀ R₁₁ R₁₂ wherein each of R₁₀, R₁₁ and R₁₂ is independently selectedfrom alkyl of 1 to about 12 carbon atoms, alkyl of 1 to about 12 carbonatoms substituted with 1 to about 25 fluorine atoms and alkenyl of 2 toabout 12 carbon atoms; or (vi) W-(CH₂)_(t) --NH--(CH₂)_(q) -- wherein tand q are independently selected integers from 2 to 6 and W is aguanidinium moiety, an amidinium moiety, --NH₂, --NHR₁₀, --NR₁₀ R₁₁, or--NR₁₀ R₁₁ R₁₂ wherein R₁₀, R₁₁ and R₁₂ are as defined herein above, andpharmaceutically acceptable salts thereof.
 2. A lipid according to claim1 wherein R₁ is an alkyl or alkenyl moiety of about 10 to about 50carbon atoms.
 3. A lipid according to claim 1 wherein R₁ is a steroidylmoiety.
 4. A lipid according to claim 3 wherein the steroidyl moiety ischolesteryl.
 5. A lipid according to claim 1 wherein R₁ is --OCH(R₆ R₇),wherein R₆ and R₇ are alkyl moieties of about 10 to about 50 carbonatoms.
 6. A lipid according to claim 1 wherein R₁ is --NH(R₈) or --N(R₈R₉), wherein R₈ and R₉ are independently an alkyl or alkenyl moiety ofabout 10 to about 50 carbon atoms.
 7. A lipid according to claim 6wherein R₁ is --N(R₈ R₉), where R₈ and R₉ are each --C₁₈ H₃₇.
 8. A lipidaccording to claim 1 wherein R₁ is a cyclic amine moiety of about 4 toabout 10 carbon atoms.
 9. A lipid according to claim 2 wherein R₁ is--C₁₈ H₃₇.
 10. A lipid according to claim 1 wherein R₂ is--N(R₄)(alk₂)-.
 11. A lipid according to claim 1 wherein R₂ is -[W₁]_(n2).
 12. A lipid according to claim 11 wherein W₁ is a substitutedamino acid residue is optionally substituted with an alkyl of 1 to about12 carbon atoms or wherein the amino group(s) is substituted to form asecondary, tertiary, or quaternary amine with an alkyl moiety of 1 toabout
 12. 13. A lipid according to claim 11 wherein W₁ is an amino acidresidue is selected from the group consisting of lysine, arginine,histidine, ornithine, tryptophane, phenylalanine, and tyrosine.
 14. Alipid according to claim 11 wherein W₁ is an amino acid analog isselected from the group consisting of 3-carboxyspermidine,5-carboxyspermidine, 6-carboxyspermine and monoalkyl, dialkyl, orperalkyl substituted derivatives which are substituted on one or moreamine nitrogens with a alkyl group of 1 to about 12 carbon atoms.
 15. Alipid according to claim 1 wherein R₂ is --NH-[alk₁ -NH]_(n1).
 16. Alipid according to claim 15 wherein alk₁ is --(CH₂)₃ --.
 17. A lipidaccording to claim 16 wherein n₁ is
 2. 18. A lipid according to claim 16wherein n₁ is
 1. 19. A lipid according to claim 15 wherein R3 isW-(CH₂)_(t) --NH--(CH₂)_(q) --.
 20. A lipid according to claim 19wherein W is --NH₂.
 21. A lipid according to claim 20 wherein t and qare each
 3. 22. A lipid according to claim 1 wherein R₃ is --NH-[alk₃]_(n3) -H, -[W₂ ]_(n4) H, or W-(CH₂)_(t) -NH--(CH₂)_(q) --.
 23. A lipidaccording to claim 1 wherein R₃ is a negatively charged group.
 24. Alipid according to claim 1 wherein R₃ is alkyl of 1 to about 12 carbonatoms optionally substituted with a substituent selected from --F, aguanidinium moiety, an amidinium moiety, --NH₂, --NHR₁₀, --N(R₁₀ R₁₁),and --N(R₁₀ R₁₁ R₁₂) wherein R₁₀, R₁₁, and R₁₂.
 25. A lipid according toclaim 1 wherein R₃ is W-(CH₂)_(t) --NH--(CH₂)_(q) --, wherein W is--NH₂.
 26. A lipid according to claim 25 wherein p and q are
 3. 27. Alipid according to claim 1 further comprising [X⁻ ] wherein X⁻ is apharmaceutically acceptable anion or polyanion and m is an integerselected such that [X⁻ ]_(m) is equal to a positive charge of the lipid.28. A lipid according to claim 1 having the structure: ##STR6##
 29. Alipid according to claim 1 having the structure:
 30. A lipid accordingto claim 1 having the structure:
 31. A composition comprising a anionicmacromolecule and a lipid according to claim
 1. 32. A compositionaccording to claim 31 wherein the anionic macromolecule comprises anexpression vector capable of expressing a polypeptide in a cell.
 33. Acomposition according to claim 31 wherein the anionic macromolecule isan oligonucleotide or an oligomer.
 34. A composition according to claim31 wherein the anionic macromolecule is DNA or RNA.
 35. A method ofdelivering an anionic macromolecule into a cell comprising contacting acomposition of claim 31 with the cell.
 36. A method to interfere withthe expression of a protein in a cell comprising contacting acomposition of claim 33 with the cell wherein the oligomer has a basesequence that is substantially complimentary to an RNA sequence in thecell that encodes the protein.
 37. A kit for delivering a anionicmacromolecule into a cell comprising a composition of claim
 31. 38. Acomposition which comprises a lipid of claim 1 and a second lipidselected from Lipid A, Lipid B and Lipid C.
 39. A composition whichcomprises a lipid of claim 19 and a second lipid selected from Lipid A,Lipid B and Lipid C.
 40. A composition which comprises a lipid of claim27 and a second lipid selected from Lipid A, Lipid B and Lipid C.
 41. Acomposition which comprises a lipid of claim 27 and Lipid A.